PIK3CA mutational status in tissue and plasma as a prognostic biomarker in HR+/HER2− breast cancer

Abstract Introduction Hotspots (HS) mutations in the PIK3CA gene may lead to poorer oncological outcomes and endocrine resistance in advanced breast cancer (BC), but their prognostic role in early‐stage disease remains controversial. The overall agreement within plasma and tissue methods has not been well explored. Our aim was to correlate tissue and plasma approaches and to analyze the prognostic impact of PIK3CA mutations (PIK3CAm) in HR+/HER2− BC. Methods A retrospective and unicentric analysis of PIK3CA mutational status in tissue and plasma samples by Cobas®PIK3CA Mutation Kit in patients with HR+/HER2− BC. Results We analyzed 225 samples from 161 patients with luminal BC. PIK3CA mutations were identified in 62 patients (38.5%), of which 39.6% were found in tissue and 11.8% in plasma. In advanced disease, plasma and tissue correlation rate was performed in 64 cases, with an overall agreement of 70.3%. Eighty patients were treated with CDK4/6 inhibitors + endocrine therapy. We observed a moderately worse progression‐free survival (PFS) in PIK3CAm versus wild‐type (WT) (24 m vs. 30 m; HR = 1.39, p = 0.26). A subanalysis was carried out based on exons 9 and 20, which showed a statistically poorer PFS in PIK3CAm exon 9 versus 20 population (9.7 m vs. 30.3 m; HR = 2.84; p = 0.024). Furthermore, detection of PIK3CAm in plasma was linked to a worse PFS vs PIK3CAm detection just in tissue (12.4 vs. 29.3; HR = 2.4; p = 0.08). Conclusions Our findings suggest the PIK3CA evaluation in tissue as the diagnostic method of choice, however, additional investigations are required to improve the role of liquid biopsy in the PIK3CA assessment. PIK3CAm show worse outcomes in advanced luminal BC, especially in exon 9 mutation carriers, despite visceral involvement, prior exposure to endocrine therapy or detection of PIK3CAm in plasma, with an unclear prognosis in early‐stage disease. Nonetheless, this should be validated in a prospective cohort study.


INTRODUCTION
2][3] Multiple investigations have revealed that this route is upregulated in approximately 60%-75% of tumors, including breast cancer (BC). 4,5ctivating mutations in the PIK3CA (PIK3CAm) gene directly alter the function of the PI3 kinase protein, causing abnormal activation of the PI3 kinase signaling pathway and promoting cancer progression.In contrast, neutral somatic mutations are common variants found in the general population that have minimal impact on protein function. 6otspot (HS) mutations in PIK3CA are detected in approximately 30%-40% of luminal BCs, 7 resulting in hyperactivation of the catalytic subunit (p110α) of phosphatidylinositol-3-kinase (PI3K).PIK3CAm most commonly occur at three HS sites: E542K and E545K in exon 9 (helical domain), and H1047R in exon 20 (kinase domain). 8he implications of PIK3CAm and patient outcomes remain controversial.Some studies have suggested poorer clinical outcomes in advanced disease, resulting in lower overall survival (OS) due to the constitutive activation of the PI3K/AKT/mTOR pathway, which promotes cell growth and invasion, and increased resistance to endocrine therapy, chemotherapy, and some targeted therapies in PIK3CAm carriers. 9,10n the other hand, PIK3CAm in early BC (eBC) has also been reported by some authors to be associated with increased recurrence-free survival 11 and responsiveness to hormonal therapy.Although its prognostic role in early stage is more debated 12,13 and may vary based on clinical circumstances and the specific treatment regimen employed.
Recently, the prognostic impact of PIK3CAm in patients treated with cyclin inhibitors has been reported, 14 identifying the presence of PIK3CAm as a promising predictive biomarker of resistance to CDK4/6 inhibitors.
Based on the phase III trial, SOLAR-1, 15 PIK3CAm have reached level 2 evidence for predicting benefit, according to the Magnitude of Clinical Benefit Scale by ESMO, from fulvestrant combined with alpelisib, an alpha-specific PI3K inhibitor in patients with advanced HR+/HER2− BC, who had previously progressed to a previous line of endocrine therapy, showing a significantly higher median PFS compared to placebo plus fulvestrant (HR = 0.65 [95% CI, 0.50-0.85],p < 0.001).Hence, with the recent approval of alpelisib for BC patients whose tumors harbor PIK3CAm, the proficiency testing for PIK3CAm analysis is essential.
In addition, plasma-derived circulating tumor DNA (ctDNA) offers real-time insight into genetic alterations and a non-invasive view of tumor heterogeneity.The determination of PIK3CAm in plasma could provide critical information and discriminate patients who would benefit from treatment with PIK3CA inhibitors.Also, the plasma detection rate of PIK3CAm in BC is highly variable, ranging from 26% to 93%, 16 and correlation rate among testing methods (tissue and plasma) has not been well explored.Our aim was to correlate tissue and plasma approaches and to analyze the prognostic impact of PIK3CA mutations (PIK3CAm) in HR+/HER2− BC.

| Study design and participants
This is a retrospective, unicentric, and observational analysis of PIK3CA mutational status in tissue and plasma in patients with HR+/HER2− BC from February 2021 to April 2023 in the University Hospital of Salamanca (HUSA).
Samples obtained from patients with HR+/HER2− BC were collected from tissue (primary tumor or metastases) and plasma.Liquid biopsy was obtained exclusively from patients with metastatic disease.The patients met the following criteria: histopathological confirmed diagnosis of BC, with complete clinical and histological data.using Cobas® DNA/cfDNA sample preparation kit (Roche Diagnostics), according to the manufacturer's guidelines.A comprehensive analysis of the technical process of tissue and plasma sample extraction is included in the Data S1.
The DNA obtained from FFPE and liquid biopsy samples were used to analyze 17 mutations across exons 1, 4, 7, 9, and 20 of the PIK3CA gene (Roche Diagnostics) using the Cobas® PIK3CA Kit (Cobas® platform, Roche Diagnostics). 17he Cobas® PIK3CA Mutation Test platform is specifically designed to differentiate activating mutations in the PIK3CA gene from neutral somatic mutations and germline polymorphisms.It focuses on recognized mutation HS within PIK3CA (associated with functional changes in the PI3 kinase protein, which can lead to abnormal activation of the PI3 kinase signaling pathway), utilizing an extensive database of validated mutations, rigorous quality control measures, and real-time PCR technology for targeted amplification and sequencing of pertinent gene regions.Detailed clinical reports are generated to provide precise information for making informed therapeutic decisions based on the mutation profile identified in the patient's PIK3CA gene. 17

| Study outcomes
Clinical assessment criteria, such as response rate, disease-free survival (DFS), or metastasis-free interval and progression-free survival (PFS) were analyzed.
We correlated both diagnostic methods (tissue and plasma) and examined the discordant cases and patient characteristics.We analyzed clinical outcomes in PIK3CAm and wild-type (WT) populations in terms of metastasis-free interval and PFS during treatment with CDK4/6 inhibitors, according to HS mutations (exon 9 vs. 20), visceral involvement, metastatic sites, and detection in plasma.

| Statistical analysis
A descriptive analysis of the clinicopathological features of the population was performed according to PIK3CA status (PIK3CAm vs. WT).To assess the quality of the statistical data for group differentiation, we used Student's t-test and Mann-Whitney U-test for normally and abnormally distributed continuous variables, respectively.SPSS v.22 (IBM Corp., Armonk, NY, USA) was applied to calculate continuous variables and measures of central tendency, such as mean and standard deviation.
Frequencies and percentages were indicated as dichotomous variables using Pearson's X 2 test.The statistical significance of the differences in survival was determined by the Mantel-Cox test (log-rank test) and the Kaplan-Meier method.Cox regression models were used to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs) for PFS.Multivariate analysis of prognostic factors for PFS was carried out using multivariate Cox regression models.Results were defined as significant when twosided p-values were <0.05.

| Mutational spectrum
We included 161 patients, 71 with eBC and 90 patients with advanced disease.A total of 225 samples from 161 patients were analyzed: 149 tissue samples and 76 plasma samples.Mutations in the PIK3CA gene (n = 69) were identified in 62 patients (38.5%).Of these, 39.6% were in tissue (59 patients) and 11.8% were in plasma (9 patients).Seven patients (11.3%) had >1 activating mutation in the PIK3CA gene.Plasma was obtained exclusively from patients with advanced disease.

| Plasma-tissue correlation analysis
We conducted a plasma-tissue correlation study in 64 metastatic patients for whom paired samples were available.We found PIK3CAm in 28 (43.8%).The overall tissue-plasma correlation rate was 70.3%, with a positive agreement in 9 of 28 patients (32.1%).Interestingly, a discordant result was observed in 19 cases (29.7%) with the detection of PIK3CAm in tissue but not in plasma, as shown in Table 1.
The timing of the samples was characterized by the median time (in days) between tissue biopsy and plasma collection.For the overall population, we observed a median interval of 779.5 days with an interquartile range (IQR) of 1150.In patients with PIK3CAm, this interval extended to 1256 days with an IQR of 2309, while for those with PIK3CA WT, it was 721 days with an IQR of 954.We found no significant association between the timing of sample collection and the presence of PIK3CAm; p = 0.54.

| Patient characteristics
Clinicopathological characteristics are listed in Table 2.One hundred sixty-one patients were included, with a median age of 54 years [21-91].Seventy-one patients with eBC and 90 patients with advanced disease were analyzed.According to histological subtype, we observed a significantly higher proportion of BC with lobular histology in PIK3CAm carriers versus WT [18.1% vs. 4.1%, OR = 5.12 (95% CI, 1.55-16.89);p = 0.007].Other clinicopathological features reflected in Table 2 showed a similar proportion between both groups (PIK3CAm vs. WT).Smaller tumors (<50 mm), less nodal involvement, lower Ki-67 index, and histological grade were observed in PIK3CAm versus WT population, although not statistically significant.

| Prognostic value of PIK3CAm
In the overall population assessed for DFS (n = 127); 78 patients were PIK3CAm (61.4%) and 49 were PIK3CA WT (38.6%), excluding those with advanced stage at onset.During follow-up, 56 patients developed metastatic disease, while 71 did not experience recurrences.
Univariate and multivariate analysis were conducted to evaluate the impact of PIK3CAm mutational status on DFS in eBC patients.Multivariate analysis in Figure 2, identified nodal involvement as an independent prognostic factor for DFS in eBC [HR = 2.76 (95% CI, 1.1-6.94);p = 0.03].
In patients undergoing treatment with cyclin inhibitors, we conducted a subanalysis focusing on PIK3CAm in exons 9 (n = 15) and 20 (n = 12), comprising a total of 27 subjects.
In 22 patients, cyclin inhibitors were used as first-line treatment, while in 5 cases, prior exposure to endocrine therapy for advanced disease was reported.Interestingly, we observed that patients with PIK3CAm on exon 9 had a significantly poorer PFS versus those with PIK3CAm on exon 20 [9.7 m vs. 30.3m; HR = 2.84 (95% CI, 1.1-7.4),p = 0.024].The results of the exon analysis are summed up in Figure 6.No significant differences were observed in response rates between PIK3CAm and WT population; [73.3% vs. 75%; OR = 0.92 (95% CI, 0.16-5.21);p = 0.92].
Even though we reported a trend to worse PFS in patients with prior exposure to endocrine therapy in advanced disease [HR = 2.73 (95% CI, 0.87-9.32);p = 0.07], Bold values indicate a significant association between PIK3CAm and the histological type of breast cancer.Notably, patients with PIK3CAm exhibit a higher prevalence of lobular histology (18.1%) compared to those without the mutation (4.1%).This difference is statistically significant, as indicated by a p-value of 0.007.

PIK3CAm
T A B L E 2 Clinical and pathological features of carriers and non-carriers of PIK3CAm in our sample.

| DISCUSSION
In our research, we found PIK3CAm in 38.5% of BC (62 patients), 77.9% occurring at the three HS (E542K and E545K at exon 9 and H1047R at exon 20).Our results agree with those previously reported: PIK3CAm rate ranges from 30% to 40% of BC 7 and is more frequent in HR+ disease.Similar mutation rates have been reported in advanced disease (between 30% and 40%) 15,18 and in the three HS mentioned above (70%).Due to the clinically significant results of PIK3CA inhibitors in metastatic BC patients 15 and the spatial heterogeneity of PIK3CA expression within BC (from primary tumor to metastases 19 ), the reliable screening for PIK3CAm is still of paramount importance, as it could guide clinicians' decision making.
We found no significant differences in the PIK3CAm status (PIK3CAm vs. WT) between eBC and metastatic disease (38.0% vs. 38.8%;p = 0.91).Similarly, no differences were reported according to the biopsy location, whether from the primary tumor or metastasis.As presented in our study, there is a similar PIK3CAm rate in primary tumors and metastases 20 and discordances are relatively low (approximately 10%).Along with that, Juric et al., 21 reported that the rate of PIK3CA WT eBC that changed to PIK3CAm is quite unusual, which could explain a stable incidence between early and late-stage BC.
According to the screening of PIK3CAm, detection in fresh tumor samples and ctDNA depends on analytical and methodological reasons, which possibly affects clinical utility validation. 22e performed a plasma and tissue correlation study in 64 metastatic patients, with an overall correlation rate of 70.3%.It is noteworthy that 19 out of 28 patients had a discordant result: PIK3CAm was detected in tissue and missing in plasma.
Limited studies have explored the agreement of ctDNA vs tissue-based methods.They show a widely variable concordance rated (26%-93%) between the two approaches 16,23,24 due to low or non-tumor shedding, technical difficulties or tumor heterogeneity.
In our study, we reported a moderate discordance rate (29%) that could be explained by the sensitivity of the technique used for ctDNA testing 25 (Cobas®PIK3CA Kit) with an analytical sensitivity of 0.7%-3.5%. 17This test provides a significantly lower detection capacity than other techniques, such as exosome-derived DNA analysis, next generation sequencing or digital PCR (detection of PIK3CAm below 0.1%).Also, observed a higher plasma correlation in patients with visceral involvement, ≥3 metastatic sites, as well as collection during disease progression, which would translate into a higher tumor burden and ctDNA. 25his is in line with what has been published by Oliveira et al., 26 who identified greater cost-effectiveness in detecting mutations in disease progression status.
In relation to the clinicopathological characteristics, we observed a significantly higher proportion of BC with lobular histology in PIK3CAm population (18.1% vs. 4.1%; p = 0.007).Nonetheless, most studies have been carried out on invasive ductal BC, and only sporadic cases of less common histologies with better prognoses have been explored for PIK3CA abnormalities. 27Based on some reports, the frequency of PIK3CAm was particularly high (up to 46% of cases) in the lobular histology, with one of the highest incidences recorded so far for different types of human neoplasms. 28lthough in our study we did not report significant differences in other clinicopathological features, we observed a trend toward smaller tumors, with less lymph node involvement and a lower Ki-67 index.This is in line with Kalinsky et al., 29 who described the association with good prognostic features in PIK3CAm eBC, including lymph node negativity (p = 0.03), lower tumor stage (p = 0.02) and lower grade (p < 0.001).
In addition, PIK3CAm seems to display differential prognostic value in eBC; as a favorable prognostic factor 29 but questionable by some authors, 30 whereas, in metastatic setting, it has been strongly associated with poorer OS and resistance to chemo and endocrine therapies in advanced disease. 18,31In the study published by Dr. Kok and colleagues, 32 no prognostic impact of somatic was observed in women with HR+/HER2− eBC on DFS and OS.In line with this, we did not observe in our population a higher DFS (time to metastases) in PIK3CAm vs WT (p = 0.94).According to our multivariate analysis, PIK3CA status did not appear to be as crucial as lymph node involvement (HR = 2.76; p = 0.03) or tumor grade (HR = 2.18; p = 0.08) in relapsing disease.
Based on the endocrine resistance conferred by PIK3CAm, we described in advanced disease worse PFS in PIK3CAm patients treated with cyclin inhibitors (21.9 m vs. 30.1 m; p = 0.08).This detrimental effect was greater in patients with visceral involvement plasma detection of PIK3CAm and in exon 9 PIK3CAm carriers.Our results are along with those presented by Del Re et al., 14 in which they describe that PIK3CAm detection in liquid biopsy correlates with a poorer PFS in population with metastatic BC undergoing treatment with CDK4/6 inhibitors (7.44 vs. 12.9 months, p = 0.01).
However, there is still controversy regarding the prognosis associated with different PIK3CAm. 12,13Our data confirm the results of Barbareschi and colleagues, 13 in which exon 9 PIK3CAm were strongly related to early recurrence and death compared with exon 20 PIK3CAm with a better prognosis.
While these studies suggest poorer outcomes of certain PIK3CAm in the pre-cyclin inhibitor era, our research supports the potential deleterious effect of mutations in exon 9 during CDK4/6 inhibitors treatment.
Therefore, this population represents an unmet clinical need.Its identification could be critical for guiding future therapeutic decisions and could be reconsidered as a potential predictive biomarker of resistance to CDK4/6 inhibitors.
In addition, a similar range of benefits has been reported with the PIK3CA inhibitor (alpelisib) in exons 9 and 20 PIK3CAm carriers, in agreement with the outcomes of the phase 3 SOLAR-1 trial, 15 representing a potential therapeutic option in this poorer prognosis population.

| CONCLUSIONS
In conclusion, our findings suggest the PIK3CA evaluation in tissue as the diagnostic method of choice, however, additional investigations are warranted to better define the role of liquid biopsy in the PIK3CA assessment.Improvements in liquid biopsy techniques for detecting PIK3CAm, combined with careful timing and selection of clinicopathological characteristics, could significantly enhance detection sensitivity rates.Despite the study's limited sample size, PIK3CAm appear to correlate with poorer outcomes in advanced luminal BC patients treated with CDK4/6 inhibitors; significantly worse in exon 9 mutation carriers, regardless of visceral involvement and plasma detection, highlighting an urgent and unmet clinical concern.In early-stage disease, PIK3CAm did not demonstrate a significant prognostic impact.These findings underscore the need for validation in a larger, prospective cohort study.

1. 2 |
Technical analysis of PIK3CA in plasma and tissue DNA extraction from formalin-fixed paraffin-embedded (FFPE) tumor tissue and liquid biopsy was conducted despite visceral involvement, prior exposure to endocrine therapy or detection of PIK3CAm in plasma, with an unclear prognosis in early-stage disease.Nonetheless, this should be validated in a prospective cohort study.K E Y W O R D S ctDNA, cyclin inhibitors, luminal breast cancer, PIK3CA, tissue | 3 of 11 TERÁN et al.

F I G U R E 1
Disease-free survival (DFS) according to the molecular status of PIK3CA gene.

F I G U R E 2
Multivariate analysis evaluating the prognostic impact in terms of DFS of clinical and molecular variables in eBC patients.F I G U R E 3Outcomes according to progression-free survival (PFS) in PIK3CAm and WT patients with cyclin inhibitor treatment.

F
I G U R E 4 Progression-free survival (PFS) in the PIK3CAm population regarding visceral involvement.F I G U R E 5 Progression-free survival (PFS) in PIK3CAm regarding PIK3CA status in ctDNA.| 9 of 11 TERÁN et al.
Tissue and plasma correlation analysis of PIK3CAm carriers.
T A B L E 1